Malignant brain tumors represent one of the most refractory cancers to therapy and remain incurable. Gliomas represent the most common type of brain tumors that occur in various grades, with the patient's prognosis inversely proportional to the grade. The long-term objective of my laboratory is to understand the cellular and molecular mechanisms that underlie tumor invasiveness in human gliomas. My laboratory has been active in the study of proteases and the biology of brain tumors, and data generated so far have indicated that changes in proteases are correlated with the changes in the grade of the tumors. The hypotheses are: 1) Regulation of cathepsin B and uPAR expression in human gliomas via a vector expressing siRNA for cathepsin B and uPAR message will inhibit tumor growth, invasion and angiogenesis and 2) Regulation of cathepsin B and uPAR protein levels by bicistronic RNAi constructs in glioma cell lines decrease the level of signaling pathway molecules which are involved in cell survival, migration, proliferation and angiogenesis. The Specific Aims to address these hypotheses are as follows: Specific Aim 1. Evaluate the effect of vectors expressing siRNA targeting cathepsin B and uPAR (pCU/AdCU) on glioma cell growth, attachment, migration and invasion in vitro, la. Determine the effect of vectors expressing siRNA against cathepsin B and uPAR (pCU/AdCU) on cathepsin B and uPAR levels in glioblastoma cell lines. Ib. Compare the effect of the pCU/AdCU constructs on glioma growth, adhesion, apoptosis and migration with that of control/mock EV (empty vector) and SV (scrambled vector) in vitro. Ic. Investigate the effect of the pCU/AdCU constructs on the invasive behavior of human glioblastoma cells in in vitro models with that of control/mock, EV (empty vector) and SV (scrambled vector). Specific Aim 2. Determine the in vivo effects of pCU/AdCU on invasion and tumorigenicity in nude mice. 2a. Access the ability of the pCU/AdCU constructs in pre-established intracranial tumor growth or invasiveness of human glioblastoma cell lines (SNB19 and U251) injected intracranially in nude mice. 2b. Determine the effect of pCU/AdCU constructs in pre-established intracranial tumor growth or invasiveness of two xenografts in nude mice. Specific Aim 3. Determine the effect of pCU/AdCU on the molecular mechanism of proliferation, apoptosis and angiogenesis. 3a. Evaluate the effect of pCU/AdCU on the molecular mechanism of proliferation. 3b. Evaluate the effect of pCU/AdCU on the molecular mechanism of apoptosis. 3c. Evaluate the effect of pCU/AdCU on cerebral angiogenesis in both in vitro and in vivo models. We anticipate that these results will substantially augment our understanding of how cathepsin B and uPAR molecules are regulated;thus, information gained should be of help in developing new therapeutic approaches to treat glioblastomas.